1. Field of the Invention
This invention relates to assaying the urea content of fluids and aqueous solutions and, more particularly, to test means and methods for improving the stability of colorimetric assays for the rapid and accurate determination of urea.
2. Description of the Prior Art
Urea is a chief end product of protein metabolism in the human body. Urea concentration in the blood accordingly provides an indicator of kidney function. Elevation of the urea concentration in blood signifies inadequate kidney function. Since toxic substances are retained in the blood in rough proportion to urea level, a high nonprotein nitrogen or blood-urea-nitrogen level is a matter of concern to a physician.
One of the commonest causes of high blood urea values (uremia) is renal disease which may be either acute or chronic. Any of the inflammatory, degenerative, congenital or neoplastic ills that affect the kidney can cause uremia, and the degree of uremia provides a rough index to the severity of the existing condition.
In practice, the concentration of urea in blood is normally expressed in terms of blood-urea-nitrogen (BUN). This BUN value represents the amount of nitrogen present as urea and is approximately one half of the total urea value. When either the urea or nitrogen value has been determined, the other value can be calculated therefrom.
The normal range of BUN values in individuals varies from between 5 and 20 milligram percent (mg%). No significance is ordinarily attached to the lower values. However, elevations in BUN values generally indicate the presence of an abnormal condition. The most common cause of increased blood-urea-nitrogen is inadequate excretion, usually due to a kidney disease or urinary obstruction. For example, in acute nephritis the BUN level may vary from 25 mg% to as high as 160 mg%. Elevated urea retention also occurs in extensive parenchymatous destruction of kidney tissue, as in pyelonephritis, advanced nephrosclerosis, renal tuberculosis, renal cortical necrosis, renal malignancy, renal suppuration or chronic gout. Although BUN values can rise to as high as 400 mg%, they usually do not exceed 200 mg%. A need thus exists in the medical field today for a rapid and accurate analytical technique for determining BUN. In the past, cumbersome wet chemical methods have been used to perform such analysis. Other methods, such as conductivity methods, spectrophotometric techniques and colorimetric techniques have also been utilized. While these methods are generally accurate, the techniques tend to be somewhat time consuming and can require careful interpretation.
In perhaps the most commonly used method for determination of urea in biological fluids, urea is hydrolyzed to ammonium carbonate by means of the enzyme urease in the presence of a buffer solution. The amount of ammonia liberated is determined colorimetrically. This first method is very specific and sensitive, but has the drawback of urease inhibition and inactivation, the problem of reagent stability and a further drawback in that the method measures endogenous ammonia. Another procedure utilizes hydrolysis with urease and requires special apparatus not always available in the routine laboratory. Yet another procedure employs the direct colorimetric reaction of urea in a protein free filtrate with an organic reagent such as diacetyl monoxime. The diacetyl reaction has the disadvantage that the color produced in unstable, the fact that colors are developed at about 95.degree. C. and that the volatile diacetyl has an odor which is unpleasant to at least some individuals.
In U.S. Pat. No. 4,074,972, assigned to American Monitor Corporation, a liquid assay for urea is disclosed which involves a reaction between a sample of biological fluid and an acidic reagent solution of o-phthalaldehyde and 8-(4-amino-1-methyl butyl amino)-6-methoxyquinoline. There is neither a teaching nor a suggestion of incorporating the reagent solution into a test means, such as a carrier matrix, let alone a strong cation exchange resin loaded carrier matrix which is in its acid form. Moreover, this patent does not disclose any method of overcoming the stability problem which occurs when the reagents of the acidic reagent solution are combined. Another problem of the disclosed assay is the fact that determinations must be made at wavelengths in the range of 470 to 540 nanometers which is close to the blank color absorption maximum resulting in the likelihood of reflectance spectroscopy measurement errors.
In the industrial area, urea is an important addition to process fluids such as plating baths, and a quick means to determine the concentration of urea therein is of great value. Urea itself is used as a fertilizer, and a quick means for assaying this material is important as a control measure in its production and use.
Accordingly, for both medical and industrial applications there is a need for assaying the urea content of fluids which can be run quickly without highly specialized equipment or trained technicians to operate such equipment.